Excretable Reporter Systems

Abstract

The present invention provides a reporter system comprising a reporter gene encoding a reporter protein that is secretable from cells in which it produced or expressed either in vitro or in vivo and excretable in a body fluid from whole animals comprising such systems. The reporter system is useful for the detection of gene activation events or biochemical changes related to, or that occur, as a result of altered metabolic or disease status or toxicological stress in toxicological screening.

1 . A nucleic acid construct comprising a nucleic acid sequence further comprising a nucleic acid sequence encoding a reporter protein that is secretable or excretable as a protein or product from a cell where the protein or product is expressed or produced.  
     
     
         2 . The nucleic acid construct according to  claim 1 , wherein the secretable/excretable protein or product is produced by modulated gene transcription.  
     
     
         3 . The nucleic acid construct according to  claim 1 , wherein the secretable/excretable protein or product is produced by increased reporter translation.  
     
     
         4 . The nucleic acid construct according to  claim 3 , wherein the increased reporter translation is a function of increased stability or decreased turnover of mRNA.  
     
     
         5 . The nucleic acid construct according to  claim 1 , wherein the secretable/excretable protein or product is produced by post-translational modulation.  
     
     
         6 . The nucleic acid construct according to  claim 5 , wherein the post-translational modulation is increased reporter stability through removal of polyubiquination or a function of accumulation or excretion of small molecule metabolites.  
     
     
         7 . The nucleic acid construct according to  claim 37 , wherein the peptide tag further comprises an epitope tag or a tag comprising enzymatic activity.  
     
     
         8 . The nucleic acid construct according to  claim 37  further comprising a promoter element upstream of (i) the nucleic acid sequence encoding the secreted/excreted protein, and/or (ii) a nucleic acid sequence encoding the peptide tag.  
     
     
         9 . The nucleic acid construct according to  claim 1 , wherein the secreted/excreted reporter protein is secreted alkaline phosphatase (SEAP).  
     
     
         10 . The nucleic acid construct according to  claim 9 , wherein the construct further comprises a CypA1 promoter.  
     
     
         11 . The nucleic acid construct according to  claim 1 , wherein the secreted/excreted reporter protein is a modified human β choriogonadotrophin (hCG) molecule.  
     
     
         12 . The nucleic acid construct according to  claim 11 , wherein the construct further comprises a stratifin gene promoter.  
     
     
         13 . The nucleic acid construct according to  claim 11 , wherein the hCG molecule is tagged.  
     
     
         14 . The nucleic acid construct according to  claim 13 , wherein the hCG molecule is myc-tagged.  
     
     
         15 . The nucleic acid construct according to  claim 1 , wherein the secreted/excreted reporter protein/product is selected from the group consisting of hormonal molecules, antibodies and enzymatic molecules.  
     
     
         16 . The nucleic acid construct according to  claim 15 , wherein the hormonal molecule is FSH.  
     
     
         17 . The nucleic acid construct according to  claim 15 , wherein the antibody is γ or light chain (Bence Jones) protein.  
     
     
         18 . The nucleic acid construct according to  claim 15 , wherein the enzymatic molecule is feline urinary carboxylase.  
     
     
         19 . A host cell comprising at least one nucleic acid construct according to  claim 1 .  
     
     
         20 . A cell line comprising at least one nucleic acid construct according to  claim 1 .  
     
     
         21 . A transgenic non-human animal wherein the cells of the non-human animal express the protein or product encoded by the nucleic acid sequence of the nucleic acid construct according to  claim 1 .  
     
     
         22 . The transgenic non-human animal according to  claim 21 , wherein the non-human animal is a mammal.  
     
     
         23 . The transgenic non-human mammal according to  claim 22 , wherein the mammal is a mouse.  
     
     
         24 . The transgenic non-human animal according to  claim 21 , wherein the secreted/excreted reporter protein or product is excreted in a body fluid selected from the group consisting of urine, saliva, tears, milk, cerebrospinal fluid and semen.  
     
     
         25 . The transgenic non-human animal according to  claim 21 , wherein the secreted/excreted reporter protein or product is excreted in urine.  
     
     
         26 . The host cell according to  claim 19 , wherein the secreted/excreted reporter protein or product has a molecular weight of about less than 60-120 kDa.  
     
     
         27 . The host cell according to  claim 19 , wherein the secreted/excreted reporter protein or product comprises a hydrophilic globular tertiary structure, low bio-activity and/or is distinguishable from endogenous molecules.  
     
     
         28 - 30 . (canceled)  
     
     
         31 . A method of detecting a gene activation event in a cell in vitro or in vivo, comprising assaying a host cell or transgenic non-human animal each comprising a nucleic acid construct according to  claim 1 , wherein the cell or animal is subjected to a gene activation event that is signalled by expression of a secreted/excreted reporter protein wherein the protein is optionally tagged with an epitope.  
     
     
         32 . A method of screening for, or monitoring of, toxicologically induced stress in a cell or a cell line or a non-human animal, comprising evaluating a cell, cell line or non human animal each comprising the nucleic acid construct according to  claim 1 .  
     
     
         33 . A method for screening and characterizing viral, bacterial, fungal, and parasitic infection or screening for cancer, inflammatory disease, cardiovascular disease, metabolic disease, neurological disease and disease with a genetic basis comprising evaluating a cell, cell line or non human animal each comprising the nucleic acid construct according to of  claim 1 .  
     
     
         34 . The nucleic acid construct according to  claim 1 , wherein the reporter protein is expressed or produced in vitro or in vivo.  
     
     
         35 . The nucleic acid construct according to  claim 1 , wherein the reporter protein is secretable/excretable from a whole animal.  
     
     
         36 . The nucleic acid construct according to  claim 35 , wherein the whole animal is a transgenic non-human animal.  
     
     
         37 . The nucleic acid construct according to  claim 1 , wherein the construct further comprises a peptide tag.  
     
     
         38 . A reporter system comprising at least two nucleic acid constructs, wherein the nucleic acid constructs each comprise: 
 (i) a nucleic acid sequence encoding a reporter protein; and    (ii) a nucleic acid sequence encoding a peptide tag to the reporter protein,    wherein each reporter protein is distinct from the proteins normally expressed in the host comprising the reporter system.    
     
     
         39 . The reporter system according to  claim 38 , wherein the at least two nucleic acid constructs encode a same reporter protein having a different peptide tag or encode a different protein having a same peptide tag.  
     
     
         40 . The reporter system according to  claim 38 , wherein the reporter protein is selected from the group consisting of secreted alkaline phosphatase (SEAP), a γ or light chain (Bence Jones) protein and feline urinary carboxylase.